The present invention relates to agents which are useful in the diagnosis and treatment of malignancies such as cancer therapy, to processes for their production and to pharmaceutical compositions containing them.
Apoptotic cell death is characterised by loss of cytoplasmic material, nuclear changes with marginalisation of chromatin and by the formation of apoptotic bodies (L. M. Schwartz et al., Immunol. Today 1993, 14:582-590, D. J. McConkey et al. Mol. Aspects Med. (1996) 17:1-115, J. F. Kerr et al., Cancer (1994) 73:2013-26). The reduction in cell viability is accompanied by DNA fragmentation that proceeds in steps with initial formation of high molecular weight (HMW) DNA fragments (50-300 kbp) and the subsequent appearance of oligonucleosome length DNA fragments consisting of oligomers of approximately 200 bp. (M. J. Arends et al., Am. J. Pathol. (1990) 136;593-608, B. Zhivotovosky et al., FEBS Lett. (1994) 352;150-4). Cytoplasmic proteases and Ca2+-dependant signaling pathways are activated prior to DNA fragmentation, and are regarded as a prerequisite for the nuclear changes (B. Zhivotovsky et al. Exp. Cell Res. (1995) 221:404-412 m S. Kurar et al., TIBS, (1995) 20:198-202). Agonists like Fas-ligand and TNF first bind to cell surface receptors and then activate transmembrane signaling events that cause cytoplasmic and nuclear changes (L. G. Zheng et al., Nature (1995) 377: 348-351, W. P. Declercq et al, Cytokine (1995, 7:701-9, T. S. Griffith, Science, (1995) 270:1189-1192). Endonuclease activation and DNA fragmentation require that signals from the cytoplasm reach the nucleus. The mechanisms of nuclear uptake and signaling across the nuclear membrane in apoptotic cells remain poorly understood. The transport of macro-molecules from the cytoplasm into the nucleus is highly regulated. Nuclear pore complexes (NPCs) are the sites of exchange of macromolecules between cytoplasm and nucleoplasm (D. A. Jans et al., Physiol. Rev (1996) 76: 651-685, D. Gorlich et al., Science (1996) 271:1513-1518, and Y. Yoneda, J. Biochem. (1997) 121:811-817). The NPCs allow passive diffusion of molecules smaller than 30 kDa but larger proteins like ovalbumin are delayed and bovine serum albumin (66 kDa) does not enter the nucleoplasm. Entry of large molecules or complexes into the nucleus requires active transport and is commonly carrier mediated. The specificity for the carrier may be determined by the so called nuclear targetting or nuclear localization sequences (NLS) that characterize proteins with the ability to enter the nucleus. For example, binding of glucocorticoids to their receptor releases HSP 90 that binds to unoccupied receptors and reveals a NLS in the glucocorticoid receptor sequence that leads to the transport of the glucocorticoid ligand-receptor complex into the nucleus (J. Yang et al., Mol. Cell. Biol. (1994) 14: 5088-98 issn 0270-7306).
A protein complex obtainable from milk that induces apoptosis in tumour cells and immature cells but spares other cells has been described previously (Proc. Natl. Acad. Sci, USA, 92, p8064-8068). The active fraction was initially isolated from human casein by ion-exchange chromatography and was shown by N-terminal amino acid sequencing and mass spectrometry to contain an oligomeric form of xcex1-lactalbumin (described as a multimeric form or xe2x80x9cMALxe2x80x9d). Monomeric xcex1-lactalbumin is the major protein component in human milk whey, where it occurs at concentrations around 2 mg/ml (W. E. Heine et al., J. Nutr. (1991) 121: 277-83), but monomeric xcex1-lactalbumin isolated from human whey did not induce apoptosis. Further analysis has provided evidence that the apoptosis-inducing fractions contains oligomeric forms of xcex1-lactalbumin with structural properties distinct from monomeric xcex1-lactalbumin as it occurs in whey. The apoptosis inducing fraction is referred to hereinafter as MAL. It is possible that the mechanism by which the oligomer induces apopotosis may relate to the Ca2+ binding properties of MAL since apoptosis required extracellular calcium.
MAL may be derived from other sources of xcex1-lactalbumin such as bovine, sheep or goats milk or human whey.
It has now been found that MAL is taken up by susceptable cells (i.e. tumour cells) and accumulated in cell nuclei. This high uptake by the nucleus, combined with its oligomeric protein structure, means that MAL would provide a useful carrier for other moieties for example, cytotoxins or chemotherapeutic agents whose effect would supplement the a effect of MAL in killing tumour cells, or diagnostic reagents such as dyes or radio- or other labels which would allow identification of tumour cells, whilst at the same time, allowing MAL to exert a killing effect on those cells.
The present invention provides an agent comprising a protein complex comprising an oligomeric form of xcex1-lactalbumin (MAL) and a further reagent which is combined with MAL such that it is carried into the nucleoplasm of cells which are susceptible to MAL.
The said further reagent may be coupled by conjugation or by covalent bonding for example by way of a linking or spacer group as would be understood in the art. Enzymatic reactions can mediate or facilitate the coupling.
Recombinant production techniques allows also the possiblity that MAL could be produced in the form of a fusion protein with the said further reagent.
Examples of said further reagents include cytoxins such as known chemotherapeutic reagents used for the treatment of cancer, microbial toxins such as diptheria toxin and monoclonal antibodies. Alternatively, the said further reagent comprises a labelling agent such as biotin or radioactive labels such as 125I. For example, a labelling group can be introduced into a protein using an enzymatic reaction or by having a labelled building stone (such as radioactive isotopes e.g. 14C, 35S, ) within the protein. 125I-labelling can be performed enzymatically by coupling 125I to the protein with the help of lactoperoxidase. Biotinylation of the protein is performed by letteing D-biotinoyl-xcex5-aminocaproic acid-N-hydroxysuccinimide ester react with the protein by forming a stable amide bond to free amino groups in the protein.
Protein may also be labelled by adding radioactive amino acid during the production of a recombinantly produced protein.
Depending upon the nature of the said further reagent, the complex of the invention can be used in the diagnosis and/or treatment of cancer. For this purpose, the complex is suitably formulated as a pharmaceutical composition and these form a further aspect of the invention.
The complex can be administered in the form of an oral mucosal dosage unit, an injectable composition, or a topical composition. In any case the protein is-normally administered together with the commonly known carriers, fillers and/or expedients, which are pharmaceutically acceptable.
In case the protein is administered in the form of a solution or cream for topical use the solution contains an emulsifying agent for the protein complex together with a diluent or cream base. Such formulations can be applied directly to the tumour, or can be inhaled in the form of a mist into the upper respiratory airways.
In oral use the protein is normally administered together with a carrier, which may be a solid, semi-solid or liquid diluent or a capsule. Usually the amount of active compound is between 0.1 to 99% by weight of the preparation, preferably between 0.5 to 20% by weight in preparations for injection and between 2 and 50% by weight in preparations for oral administration.
In pharmaceutical preparations containing complex in the form of dosage units for oral administration the compound may be mixed with a solid, pulverulent carrier, as e.g. with lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, amylopectin, cellulose derivatives or gelatine, as well as with an antifriction agent, such as magnesium stearate, calcium stearate, polyethylene glycol waxes or the like, and be pressed into tablets. Multiple-unit-dosage granules can be prepared as well. Tablets and granules of the above cores can be coated with concentrated solutions of sugar, etc. The cores can also be coated with polymers which change the dissolution rate in the gastrointestinal tract, such as anionic polymers having a pka of above 5.5. Such polymers are hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, and polymers sold under the trade mark Eudragit S100 and L100.
In preparation of gelatine capsules these can be soft or hard. In the former case the active compound is mixed with oil, and the latter case the multiple-unit-dosage granules are filled therein.
Liquid preparations for oral administration can be present in the form of syrups or suspensions, e.g., solutions containing from about 0.2% by weight to about 20% by weight of the active compound disclosed, and glycerol and propylene glycol. If desired, such preparations can contain colouring agents, flavouring agents, saccharine, and carboxymethyl cellulose as a thickening agent.
The daily dose of the active compound varies and is dependant on the type of administrative route, but as a general rule it is 1 to 100 mg/dose of active compound at personal administration, and 2 to 200 mg/dose in topical administration. The number of applications per 24 hours depend of the administration route, but may vary, e.g. in the case of a topical application in the nose from 3 to 8 times per 24 hours, i.e., depending on the flow of phlegm produced by the body treated in therapeutic use.
The invention further provides a method for treating cancer which comprises administering to cancer cells a complex or a composition as described above.
Diagnostic applications of the complex of the invention may be carried out in vivo or in vitro for example on biopsy samples. For this purpose, a complex comprising a label may be applied to the suspect tumour in the form of a pharmaceutical composition when used in vivo or any formulation when used in vitro. The tumour can then be observed in order to determine whether the complex penetrates into the nucleus or not. Visibility of the nucleus would be indicative that the complex has been absorbed into the nucleus and is a MAL susceptible tumour. Although the degree of uptake of MAL is variable, it is taken up by cancer cells generally and therefore may be used in killing those cells, particularly when combined with another cellular toxin in a complex of the invention. Uptake of MAL is particularly high in lymphoid tumour cells such as leukaemia cells. Even in carcinoma cells such as lung cancer cells, there is sufficient uptake to result in cell death as will be illustrated hereinafter. The information obtained using diagnostic methods of the invention may assist in determining a future treatment regime.
The interaction of MAL with different cellular components was studied by confocal microscopy, using biotinylated MAL, and by subcellular factionation using 125I-labelled MAL. Monomeric xcex1-lactalbumin and human IgG were used as controls. MAL was found to accumulate in cell nuclei rather than the cytosol, the vesicular fraction or the ER-Golgi complex. The nuclear accumulation of MAL occurred rapidly in cells that were susceptible to its apoptosis-inducing effects, but not in resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of apoptosis, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from apoptosis. Ca2+ was required for MAL induced DNA fragmentation but nuclear uptake of MAL was independent of Ca2+.
The results demonstrated that MAL can target cell nuclei and that nuclear targeting mechanisms are more readily available in cells that are sensitive to MAL-induced apoptosis than in resistant cells. It appears that apoptosis induction occurs at least in part through a direct effect of MAL at the nuclear level.
Cell surface binding of MAL as a possible decisive step in apoptosis induction was investigated first. Exogenous apoptosis-inducing molecules like Fas-ligand or TNF bind to their respective cell surface receptors and trigger transmembrane signalling event and intracellular pathways leading to apoptosis. MAL bound quickly to cell surfaces, was saturate at high MAL concentrations and was specific as defined by competition experiments where labelled MAL was competed out by unlabeled MAL. By confocal microscopy MAL was ,shown to bind in patches, suggested that either MAL bound as preformed aggregates, or that the bound MAL accumulated in certain areas of the membrane through capping or other mechanisms influencing receptor distribution. There was little quantitative difference in cell surface binding of monomeric, inactive and oligomeric, active forms of the protein. Furthermore, there was no difference in cell surface binding to sensitive and resistant cells. The results suggested that MAL differs from agonists like TNF and Fas-ligand in that cell surface binding does not itself trigger apoptosis. Recently Sheridan et al. (Science, (1997) 277:818-821 and Pan et al. (Science (19970 277:815-818) described a decoy receptor lacking the signalling domain of the native receptor, in the membrane of healthy cells [Pan, 1997 #640,; Sheridan, 1997 #639]. The TRAIL protein binds cell surface receptors with similar affinity, but will not be able to induce an apoptosis-signal in healthy cells.
MAL was rapidly taken into the nuclei of cells that were sensitive to its apoptosis inducing effect, suggesting that it was capable of nuclear targeting. This term is used herein to describe preferential localisation of certain molecules to the nuclear compartment. Molecules of diverse origin, structure and function share the ability to reach cell nuclei, and may exert their main functions there as opposed to the cytoplasmic compartment. The uptake of MAL into the nucleus was via the nuclear pore complex as shown by inhibition studies using WGA, a lectin that binds to glycosylated regions of the nucleoporins and sterically hinders transport of the importin-protein complex through the nuclear pore (S. A. Adam et al., (1990) J. Cell Biol. 111:807-816). WGA treatment blocked MAL uptake into the nuclei of digitonin-treated cells and inhibited the MAL-induced DNA fragmentation. The structural basis for and mechanism of nuclear uptake of MAL need to be identified. Classical nuclear targeting sequences often include clusters of basic amino acids, that share little or no sequence homology (Jans et al. (1996) supra., J. Garcia-Bustos et al., Biochim Biophys. Acta. (1991) 1071: 83-101). Sequence analysis of the monomeric form of xcex1-lactalbumin did not show the presence of known nuclear targeting motifs and the monomer did not target cell nuclei. It is likely, therefore that MAL carried structural modifications that confer affinity for the nuclear compartment, the nuclear membrane and/or the nuclear pore.
The susceptibility to MAL-induced apoptosis in difference cells was proportional to the nuclear accumulation of MAL. MAL rapidly entered the nuclei of the sensitive L1210 cells. At a concentration of 0.3 mg/ml nuclear staining was observed after 1 hour in 10% of cells and increased to 75% after 6 hours. DNA fragmentation was first seen after 6 hours incubation. Nuclear uptake occurred more slowly in the intermediary sensitive A549 cell line and was low or absent in human kidney cells.
The difference was not observed when the total, cell-associated MAL or cytoplasmic uptake of MAL was compared between the cells. Uptake into the cytoplasm occurred with similar kinetics in the L1210, A549 and HRTEC cells. The total amount of intracellular MAL was highest in the L1210 cells but most of this was in the nuclei and not in the cytoplasm. This suggested that the nuclear uptake was the decisive step. Further evidence for a direct effect of MAL at the nuclear level was obtained using isolated nuclei. MAL induced DNA fragmentation in isolated nuclei at concentrations lower than those required for whole cells. It should be noted that isolated nuclei from the three cell types were all susceptible to the effects of MAL and showed similar kinetics of DNA fragmentation. (HMW-oligonucleosome length fragments. Differences between intact nuclei and isolated nuclei). These results suggested that the decisive event separating sensitive from resistant cells was the actual transport of MAL from the cytoplasm into the nuclei, rather than the effect of MAL once in the nuclear compartment.
Lactalbumins are Ca2+ binding proteins with one high affinity and one low affinity Ca2+ binding site (J. Ren et al., J. Biol. Chem. (1992) 268;19292-8). MAL induced apoptosis was previously shown to require extracellular Ca2+ (A. Hikansson et al., (1995) supra.). In this study, the applicants examined the effect on nuclear targeting and on DNA fragmentation of various agents that alter extra- or intracellular Ca2+ levels. Pre-treatment of sensitive cells with Ca2+-chelators was found to inhibit MAL-induced DNA fragmentation in isolated nuclei. There was however no effect on the nuclear targetting process. These observations suggested that apoptosis occurs as a result of two converging mechanisms; the transport of MAL to the nucleus and changes in the available Ca2+ concentration.
Therefore, when applying the treatment of the invention for example by using a pharmaceutical composition, care should be taken that the calcium levels are sufficient to ensure that cells can be killed. However high calcium levels can lead to inactivation of MAL. Therefore, the inclusion of calcium agents in the formulation should be avoided.
MAL contains as its major constituent oligomers of xcex1-lactalbumin. The xcex1-lactalbumin protein family has been extensively studied and characterised at the molecular level. Lactalbumins from different species show little structural variation, but the amounts are higher in e.g. human compared with bovine milk. The monomeric form of xcex1-lactalbumin has a molecular mass of 14 kDa and is the quantitatively dominating whey protein in human milk (W. E. Heine et al., (1991)supra.). MAL, on the other hand, was not derived from whey but from casein fraction of human milk after precipitation at low pH. The active fraction bound with high affinity to an ion-exchange matrix, eluted with high salt and was found to contain several oligomeric forms of xcex1-lactalbumin. Low pH and variable anionic conditions have previously been shown to alter the molecular structure of monomeric xcex1-lactalbumin to the so-called molten globule state, Molten globules are partially unfolded intermediates between the native and fully denatured forms of xcex1-lactalbumin (K. Kuwajima., Faseb J. (1996) 10:102-109, A. Alexandrescu et al., Biochemistry (1993) 32: 1707-1718). Preliminary results from structural studies suggest that MAL contains partly refolded oligomers with structural features distinct from the monomeric xcex1-lactalbumin as it occurs in human whey.
Additionally MAL often contains lipids and in particular MAL derived from human milk contains phospholipids, monoglycerides, diglycerides, cholesterol, triglycerides and free fatty acids. Specifically the free fatty acid content of MAL appears to be higher than that found in fresh milk. The role of these lipids in apoptosis or in the stabilisation of the MAL structure is not fully clear. However, the presence of these components in MAL is preferred for its therapeutic effect.
This study confirmed the difference in apoptosis inducing activity between MAL and monomeric xcex1-lactalbumin, and showed distinct difference in their cellular interactions. Monomeric xcex1-lactalbumin bound to cell surfaces and entered the cytoplasm, but did not accumulate in cell nuclei. There was no effect of xcex1-lactalbumin at the nuclear level even in digitonin-permeabilized cells, when the protein was allowed to diffuse freely into the nuclei. Furthermore, these was no difference in subcellular distribution of the monomer between apoptosis sensitive and resistant cells. The results demonstrate that structural modifications or additional milk constituents present in MAL are required for nuclear targeting and induction of apoptosis.
Human milk provides the breast-fed infant with a mucosal immune system. Molecules in milk prevent microbial attachment to mucosal tissues, lyse viral particles, disrupt bacterial cell walls and prevent microbial growth (H. McKenzie et al., Adv. Protein Chem., (1994) 44: 173-313, J. J Kabara et al., Antimicrob. Agents Chemother (1972) 2:23-28, F. D. Gillin et al., Science (1983) 221: 1290-1292). Epidemiological studies consistently find lower frequencies of viral and bacterial infections in breast-fed infants. Epidemiological studies have also provided compelling evidence that breast30 feeding may protect against cancer. Breast-fed individuals have a lower incidence of lymphomas and other malignancies, and the frequency decreases with the length of breast-feeding (M. K. Davis et al., Lancet (1988) ii: 365-368). There are other reports to suggest that the breast cancer incidence is reduced in women who breast-feed their children (V. Siskind et al., Am. J. Epidemiol. (1989) 130: 229-236, P. A. Newcomb et al., N. Engl. J. Med. (1994) 330: 81-87) Our studies provide a potential mechanism for the reduced disease frequencies. MAL may reach the rapidly proliferating cells in the gut of the breast-fed infant and drive selection through maturity and away from the neoplasia or reach the mucosa-associated lymphoid tissue and influence the function of local lymphocyte populations.
The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which: